This invention relates to proteins and polypeptides of the Bordetella outer membrane protein called pertactin and the polynucleotides that encode them. This invention also relates to the use of these proteins and polypeptides in immunogenic compositions, diagnostic methods, and diagnostic kits.
The genus Bordetella includes seven species. The most studied species are B. pertussis, B. parapertussis, and B. bronchiseptica. B. pertussis is responsible for respiratory infections only in humans. B. parapertussis causes infections in humans and sheep, and B. bronchiseptica infects many animal species, including humans.
These pathogens produce an array of virulence factors, the synthesis of which is regulated by the two-component, bvg AS (2, 21) system. These factors include toxins, such as pertussis toxin, which is the only toxin specific to B. pertussis, tracheal cytotoxin, adenylate cyclase-hemolysin, and adhesins, such as filamentous hemagglutinin, fimbriae, and pertactin (PRN).
PRN is an outer membrane protein with an apparent molecular weight of 69 kDa in B. pertussis, 70 kDa in B. parapertussis, and 68 kDa in B. bronchiseptica (5, 14, 15). The precursors of PRN are 91.5 kDa, 93 kDa, and 92.5 kDa in size, respectively. In B. pertussis, PRN has been demonstrated to be an agglutinogen (4), promoting attachment to certain eukaryotic cells via an Arg-Gly-Asp (RGD) motif (13).
Antibodies specific for the B. bronchiseptica PRN are detected at high titer in immunized piglets, whereas few if any of these antibodies are detected in unprotected animals (19). Synthesis of the PRN by B. bronchiseptica correlates with protection (16). The immunization of mice or piglets with preparations of the PRN induces protective immunity against B. bronchiseptica infection (12, 19) and passively administered monoclonal antibodies prevent the death of animals challenged with B. bronchiseptica (16). B. pertussis PRN has also been shown to induce protective immunity to intracerebral, aerosol and intranasal challenges with B. pertussis in mice (11, 18, 20).
PRN is, therefore, now included in some acellular pertussis vaccines (i.e. vaccines composed of purified bacterial proteins) (9). However, the PRN proteins of these three species, although clearly related, have different immunogenic properties. For example, preparations of B. pertussis PRN protect mice against intanasal B. pertussis challenge but not against intranasal B. parapertussis challenge (11). They also protect mice against intracerebral B. pertussis challenge, whereas the B. bronchiseptica PRN protein does not (18).
Comparison of the deduced amino acids of the three proteins, B. pertussis-PRN, B. parapertussis-PRN, and B. bronchiseptica-PRN, reveals a high degree of similarity, with the B. bronchiseptica and B. parapertussis proteins being more similar to each other than to the B. pertussis PRN protein (5, 14, 15).
The sequences of the three proteins differ in the number of repeats in regions I and II (FIG. 1a). Using monoclonal antibodies, Charles et al., identified and characterized a protective immunodominant epitope of the P.69-PRN protein (6). This epitope spans the (Pro-Gln-Pro)5 (SEQ ID NO: 30) repeat sequences located in region II. Differences in this region may account for the observation that sera from piglets that recognize B. bronchiseptica PRN do not react with B. pertussis PRN despite the high degree of similarity between these proteins (12) and for the lack of cross protection provided by the three proteins (11, 18, 20).
It has recently been shown that the PRN produced by clinical isolates of B. pertussis varies. Sequences of the prn gene of various clinical isolates revealed three major types of PRN variant (17). It has been suggested that epidemics in the Netherlands result from changes in the sequences of the genes encoding PRN and PT because the proteins present in the clinical isolates currently in circulation differ in sequence from those observed by the vaccinal strains used in this country (17).
For PRN of B. pertussis, all the observed amino acid differences are located in region I. The allelic prn types A=1 and C=3 are very similar, differing by only two amino acids, whereas type B=2 is quite different, having a five-amino acid insertion in the same region (17).
Only one type was found to differ in region II. This type (A*=6) is produced by the B. pertussis WHO reference strain 18323 and one French clinical isolate (3). It does not, however, seem to be common because it has been detected in only one clinical isolate (3). The production by this B. pertussis strain of this unusual type of PRN reflects the many common properties shared with the B. parapertussis and B. bronchiseptica species. No differences were found in the phenotype and behavior in the animal model of B. pertussis clinical isolates with different PRN (3).
There is a need in the art for compositions containing proteins and polypeptides of Bordetella pertactins that can be used in immunogenic compositions to protect against Bordetella infection and to treat subjects infected with Bordetella. Ideally, the proteins, polypeptides, and the polynucleotides that encode them would also be useful in diagnosing Bordetella infection and in kits for the diagnosis of such infection.